Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compounds

ABSTRACT With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson’s disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics. IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.

In my opinion this is an interesting study, whose key findings are adequately supported by robust experimental data.
I have only few concerns that are detailed below: 1) Besides benserazide and levodopa, the identity of the additional 18 compounds identified as PilB inhibitors in the HTS campaign should be revealed.
2) The rational behind the HTS system used in this study, that is based on the malachite green ATPase assay, should be clearly explained in the first chapter of the Results section.
3) The experiments performed to assess the specificity of benserazide and levodopa on S motility in M. xanthus are not clear to me. The rational of these experiments is that in M. xanthus S motility is dependent on T4P and can be investigated on soft agar plates, while type A motility is T4P-indepednent and can be investigated on hard agar plates. Since the inhibitors did not affect type A motility in a M. xanthus mutant with pilB deletion (DK10416) on hard agar plates, the Authors conclude that benserazide and levodopa are specific inhibitors of T4P assembly and T4P-dependent motility (i.e. S motility). However, benserazide and levodopa also decreased motility of two M. xanthus pilB-proficient strains of on hard agar plates, in a similar manner as previously observed on soft agar plates. Are T4P also required for motility on hard agar plates? Do hard agar plates allow investigating type A motility only, or both A and S type motility contribute to M. xanthus motility on hard agar plates? Please clarify. 4) In my opinion the title of the manuscript ("Discovery of two inhibitors of the type IV pilus assembly ATPase as antivirulence") does not conceive the main finding of this study, i.e. benserazide and levopoda inhibit the type IV pilus assembly ATPase PilB. Please consider that no direct evidences of the antivirulence activity of benserazide and levopoda have been produced in this study.

Minor comments
1) The acronym ABR is used only one time along the manuscript in place of antibiotic resistance (line 335). Please consider using antibiotic resistance also at line 335 and removing ABR.
2) Line 72: "pilT mutants in are hyperpiliated", please correct. Title is appropriate Rationale for the screen and execution of that screen are both sound. The use of Chloro thermophilum PilB is consistent with a previous publication in mSphere (2021) Relevance of the results for curing infections in animals and ultimately humans would require much more work beyond the scope of this paper. Do not use red and green dots because those are not distinguishable for the most common forms of color blindness. Replace one of the dots with open (white) symbols and fill one of them with a pattern such as a stripe, or change the shape of one of the symbols to something else such as a triangle . It is good that the color scheme is conserved across figures.  : I think it is odd to investigate the drugs' impact on Mx when Mx is not a pathogen, but their rationale for doing so is logical -I just would have preferred other assays using a pathogen instead. The subsequent use of Ano is more directly relevant to the ultimate goal of doing this study. One minor concern: the drugs had no effect on the growth of Mx, Ano, or Eco in broth -but these drugs would presumably have stronger effects on biofilm formation. So, the measurement of growth in broth used to argue that this drug is less likely to lead to resolution is somewhat suspect.
The paper would be enhanced by using various concentrations of substrate in the presence of the drugs to establish the change in quantitative enzyme characteristics (needed to argue that the drugs are competitive inhibitors).

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.

Response to Reviewers
We would like to thank both reviewers for your kind and constructive review of our manuscript. We greatly appreciate your expertise, time and efforts in providing timely and valuable comments and feedbacks on our manuscript. The peer review process would not work as intended otherwise.
Reviewer #1 (Comments for the Author): In this manuscript the Authors report the identification of two PilB inhibitors, benserazide and levopoda, by a high throughput screen (HTS) in vitro, and their validation as effective inhibitors of type 4 pili (T4P) assembly in vivo. Notably, benserazide and levopoda inhibit T4P-dependent phenotypes, including different types of motility and biofilm formation, in Myxoccocus xanthus and Acinetobacter nosocomialis.
In my opinion this is an interesting study, whose key findings are adequately supported by robust experimental data.
I have only few concerns that are detailed below: 1) Besides benserazide and levodopa, the identity of the additional 18 compounds identified as PilB inhibitors in the HTS campaign should be revealed.
Response: Judging by their chemical structures, some of 18 compounds are likely Pan-assay interference compounds known as PAINS in HTS-based drug discovery. A few others were found to be false positives which are also common in HTS. The remainders have either not been confirmed or not analyzed in any detail to be considered true PilB inhibitors with confidence. As a result, we feel it is better not to list the identities of these compounds to avoid disseminating misleading information.
2) The rational behind the HTS system used in this study, that is based on the malachite green ATPase assay, should be clearly explained in the first chapter of the Results section.
Response: We added the following as the 2 nd sentence in the first paragraph of the Results: "In this assay, phosphates from ATP hydrolysis and the MG reagents form green complexes that can be quantified calorimetrically with a plate reader". We also provided additional information on the HTS in the following paragraph and provided the average Z' value for the screen.
3) The experiments performed to assess the specificity of benserazide and levodopa on S motility in M. xanthus are not clear to me. The rational of these experiments is that in M. xanthus S motility is dependent on T4P and can be investigated on soft agar plates, while type A motility is T4P-indepednent and can be investigated on hard agar plates. Since the inhibitors did not affect type A motility in a M. xanthus mutant with pilB deletion (DK10416) on hard agar plates, the Authors conclude that benserazide and levodopa are specific inhibitors of T4P assembly and T4P-dependent motility (i.e. S motility). However, benserazide and levodopa also decreased motility of two M. xanthus pilB-proficient strains of on hard agar plates, in a similar manner as previously observed on soft agar plates. Are T4P also required for motility on hard agar plates? Do hard agar plates allow investigating type A motility only, or both A and S type motility contribute to M. xanthus motility on hard agar plates? Please clarify.
Response: We have added information for clarification on line 231-234 to indicate that both A and S motilities are functional on hard agar plates. This is an important and thank you for pointing it out. 4) In my opinion the title of the manuscript ("Discovery of two inhibitors of the type IV pilus assembly ATPase as antivirulence") does not conceive the main finding of this study, i.e. benserazide and levopoda inhibit the type IV pilus assembly ATPase PilB. Please consider that no direct evidences of the antivirulence activity of benserazide and levopoda have been produced in this study.
Response: We have inserted PilB in the title as we agree that it is helpful. We have left the remainder of the title as is considering the comment by the 2 nd reviewers that the "Title is appropriate".

Minor comments
1) The acronym ABR is used only one time along the manuscript in place of antibiotic resistance (line 335). Please consider using antibiotic resistance also at line 335 and removing ABR.
2) Line 72: "pilT mutants in are hyperpiliated", please correct. Response: All the above typo/grammatical errors have been corrected.

Reviewer #2 (Comments for the Author):
Title is appropriate Rationale for the screen and execution of that screen are both sound. The use of Chloro thermophilum PilB is consistent with a previous publication in mSphere (2021) Relevance of the results for curing infections in animals and ultimately humans would require much more work beyond the scope of this paper.   Response: We lowered the X intercept with Y so that the labeling is no longer obstructed by the graph. Response: We appreciate the recognition of the logic for using Mx as a model organism. As noted by the reviewer, we used the pathogen A. nosocomialis in experiments described later in the manuscript as the work progressed along. One minor concern: the drugs had no effect on the growth of Mx, Ano, or Eco in broth -but these drugs would presumably have stronger effects on biofilm formation. So, the measurement of growth in broth used to argue that this drug is less likely to lead to resolution is somewhat suspect.
Response: The experiments were to examine if these drugs have any antimicrobial activity. Broth cultures are typically used to determine the minimum inhibitory concentration (MIC) of antimicrobials.
The paper would be enhanced by using various concentrations of substrate in the presence of the drugs to establish the change in quantitative enzyme characteristics (needed to argue that the drugs are competitive inhibitors).
Response: We agree that understanding the mechanisms of inhibition by these drugs are important. We consider that the determination of the precise mechanisms is beyond the scope of this manuscript. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper soon. If your manuscript was reviewed, you will find the reviewers' comments below.
My Editor's comment to your revised manuscript is: I agree with comment 4 of Reviewer 1, as no experimental data are provided to confirm virulence inhibition. This issue should be addressed by adequate cellular and animal models, that exceed the scope of this manuscript. Therefore, I recommend more caution in the title, that should be slightly modified as follows: "Discovery of two inhibitors of the type IV pilus assembly ATPase PilB as potential antivirulence compounds". Please, notice that the acceptance of the title by Reviewer 2 does not imply that the comment of Reviewer 1 is inconsistent.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed instructions on submitting your revised paper are below.

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Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. • Manuscript: A .DOC version of the revised manuscript For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
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Thank you for submitting your paper to Microbiology Spectrum.

Response to Reviewers
We would like to thank both reviewers for your kind and constructive review of our manuscript. We greatly appreciate your expertise, time and efforts in providing timely and valuable comments and feedbacks on our manuscript. The peer review process would not work as intended otherwise.
Reviewer #1 (Comments for the Author): In this manuscript the Authors report the identification of two PilB inhibitors, benserazide and levopoda, by a high throughput screen (HTS) in vitro, and their validation as effective inhibitors of type 4 pili (T4P) assembly in vivo. Notably, benserazide and levopoda inhibit T4P-dependent phenotypes, including different types of motility and biofilm formation, in Myxoccocus xanthus and Acinetobacter nosocomialis.
In my opinion this is an interesting study, whose key findings are adequately supported by robust experimental data.
I have only few concerns that are detailed below: 1) Besides benserazide and levodopa, the identity of the additional 18 compounds identified as PilB inhibitors in the HTS campaign should be revealed.
Response: Judging by their chemical structures, some of 18 compounds are likely Pan-assay interference compounds known as PAINS in HTS-based drug discovery. A few others were found to be false positives which are also common in HTS. The remainders have either not been confirmed or not analyzed in any detail to be considered true PilB inhibitors with confidence. As a result, we feel it is better not to list the identities of these compounds to avoid disseminating misleading information.
2) The rational behind the HTS system used in this study, that is based on the malachite green ATPase assay, should be clearly explained in the first chapter of the Results section.
Response: We added the following as the 2 nd sentence in the first paragraph of the Results: "In this assay, phosphates from ATP hydrolysis and the MG reagents form green complexes that can be quantified calorimetrically with a plate reader". We also provided additional information on the HTS in the following paragraph and provided the average Z' value for the screen.
3) The experiments performed to assess the specificity of benserazide and levodopa on S motility in M. xanthus are not clear to me. The rational of these experiments is that in M. xanthus S motility is dependent on T4P and can be investigated on soft agar plates, while type A motility is T4P-indepednent and can be investigated on hard agar plates. Since the inhibitors did not affect type A motility in a M. xanthus mutant with pilB deletion (DK10416) on hard agar plates, the Authors conclude that benserazide and levodopa are specific inhibitors of T4P assembly and T4P-dependent motility (i.e. S motility). However, benserazide and levodopa also decreased motility of two M. xanthus pilB-proficient strains of on hard agar plates, in a similar manner as previously observed on soft agar plates. Are T4P also required for motility on hard agar plates? Do hard agar plates allow investigating type A motility only, or both A and S type motility contribute to M. xanthus motility on hard agar plates? Please clarify.
Response: We have added information for clarification on line 231-234 to indicate that both A and S motilities are functional on hard agar plates. This is an important and thank you for pointing it out. 4) In my opinion the title of the manuscript ("Discovery of two inhibitors of the type IV pilus assembly ATPase as antivirulence") does not conceive the main finding of this study, i.e. benserazide and levopoda inhibit the type IV pilus assembly ATPase PilB. Please consider that no direct evidences of the antivirulence activity of benserazide and levopoda have been produced in this study.
Response: We have inserted PilB in the title as we agree that it is helpful. We have left the remainder of the title as is considering the comment by the 2 nd reviewers that the "Title is appropriate".

Minor comments
1) The acronym ABR is used only one time along the manuscript in place of antibiotic resistance (line 335). Please consider using antibiotic resistance also at line 335 and removing ABR.
Response: All the above typo/grammatical errors have been corrected.

Reviewer #2 (Comments for the Author):
Title is appropriate Rationale for the screen and execution of that screen are both sound. The use of Chloro thermophilum PilB is consistent with a previous publication in mSphere (2021) Relevance of the results for curing infections in animals and ultimately humans would require much more work beyond the scope of this paper.  Do not use red and green dots because those are not distinguishable for the most common forms of color blindness. Replace one of the dots with open (white) symbols and fill one of them with a pattern such as a stripe, or change the shape of one of the symbols to something else such as a triangle . It is good that the color scheme is conserved across figures.
Response: We replaced the green color so that a colorblind person could tell the different of the three color in all the figures. Response: We lowered the X intercept with Y so that the labeling is no longer obstructed by the graph. : I think it is odd to investigate the drugs' impact on Mx when Mx is not a pathogen, but their rationale for doing so is logical -I just would have preferred other assays using a pathogen instead. The subsequent use of Ano is more directly relevant to the ultimate goal of doing this study.
Response: We appreciate the recognition of the logic for using Mx as a model organism. As noted by the reviewer, we used the pathogen A. nosocomialis in experiments described later in the manuscript as the work progressed along. One minor concern: the drugs had no effect on the growth of Mx, Ano, or Eco in broth -but these drugs would presumably have stronger effects on biofilm formation. So, the measurement of growth in broth used to argue that this drug is less likely to lead to resolution is somewhat suspect.
Response: The experiments were to examine if these drugs have any antimicrobial activity. Broth cultures are typically used to determine the minimum inhibitory concentration (MIC) of antimicrobials.
The paper would be enhanced by using various concentrations of substrate in the presence of the drugs to establish the change in quantitative enzyme characteristics (needed to argue that the drugs are competitive inhibitors).